Bone is a dynamic organ that turns over continually through bone resorption and bone formation. This remodeling process functions to maintain calcium balance, repair bone damaged from mechanical stresses, adjust for changes in mechanical load, and remove old bone material that has degraded with age. Bone mass is regulated by a delicate balance between bone resorption mediated by osteoclasts and bone formation mediated by osteoblasts.
Osteoblasts are cells of mesenchymal origin and synthesize the precursors that form the organic extracellular matrix, also called the osteoid or ground substance, which are composed mainly of type I collagen and various non-collagen proteins such as osteocalcin, osteopontin, osteonectin, proteoglycans, and alkaline phosphatases. Once a layer of organic matrix is laid down by the osteoblasts, mineralization occurs through deposition of hydroxyapatite along and within the organic matrix. Osteocalcin, a protein produced by the osteoblasts, binds and concentrates the calcium in the matrix. Consecutive layers of organic matrix added by the osteoblasts through cycles of osteoid secretion and mineralization (appositional growth) form sheets or rings of mineralized matrix, which fuse together to form a lattice structure of connected bone. A proportion of osteoblasts becomes trapped as osteocytes in the lacunae, which is connected by a system of canaliculi. In some conditions, such as in the fetus and certain bone disorders, the organic matrix is arranged in a weave-like form and results in a type of bone referred to as woven, immature, or primitive bone. Changes to stiffness of bone occurs by modulating the level of hydroxyapatite in the matrix, with higher mineral content providing stiffness and rigidity and a lower mineral content providing bone flexibility.
Osteoclasts, the primary cells responsible for bone resorption, arise from hematopoietic cells of the macrophage/monocyte lineage and are multinucleated cells {i.e., polykaryons) that form by fusion of monocytes. Osteoclasts secrete various enzymes that act in dissolution of bone material. For example, tartrate resistant acid phosphatase (TRACP) decalcifies the bone while cathepsin K digests the bone matrix proteins. Osteoclasts also acidify the surrounding environment through vacuolar H+-ATPase activity, thereby further promoting bone resorption.
The development and function of osteoclasts are tightly coupled to the activity of osteoblasts, which secrete cellular factors affecting osteoclast differentiation and activity. The osteoblast protein RANKL (receptor for activating NFkB ligand) is a key regulator that stimulates differentiation of osteoclast precursor cells and activates mature osteoclasts. Osteoblasts also produce a decoy ligand, osteoprotegrin (OPG), which competes with RANKL and inhibits its activity. Expression of RANKL is regulated by cytokines (e.g., IL-I, IL-6, IL-11 and TNF-alpha), glucocorticoids, and parathyroid hormone (PTH). The presence of RANKL upregulators leads to enhanced bone resorption and a corresponding loss of bone mass. OPG production is upregulated by cytokines IL-I and TNF-alpha, steroid hormone beta-estradiol, and mechanical stress, thereby stimulating bone formation. In contrast, glucocorticoids, and prostaglandins suppress production of OPG and thus enhance bone resorption. This intricate interaction between the osteoblasts and osteoclasts provides a mechanism for adapting to conditions requiring additional bone mass (e.g., increased mechanical load) as well as maintenance of bone mass.
Current treatments for bone loss diseases include antiresorptive agents such as bisphosphonates, calcitonin, estrogen, and vitamin D supplementation, which limit bone resorption and prevent loss of bone mass. Anabolic agents that promote bone formation have also been studied, with PTH peptide teriparatide being the only FDA approved anabolic agent. Thus there is a need for development of novel anabolic drugs to be used for treatment of bone loss diseases and/or prevention of bone loss. Moreover, therapies specifically directed against the cellular basis of bone metabolism and remodeling may avoid some of the undesirable side effects associated with some current treatments.
The abnormal regulation of osteoclast and osteoblast activities can lead to various bone disorders. The clinical presentations of decreased bone formation and/or increased bone resorption include loss of bone mass and/or decrease in structural integrity of the bone matrix. Both conditions can lead to an increased risk of bone fractures. The most common form of boneloss, primary osteoporosis, is a significant health problem because nearly 5 to 20% of the human female population suffers from the condition. Although not as prevalent as in the female population, age-related osteoporosis also affects a significant percentage of males.
Bone possesses the intrinsic capacity for regeneration as part of the repair process in response to injury. However, there are cases of fracture in which bone regeneration is impaired. For example up to 13% of fractures that occur in the tibia are associated with delayed healing or non-healing fractures. There are currently a number of treatment methods available which can be used either alone or in combination, for management of these complex clinical situations.
The tissue-engineering approach is a promising strategy added in the field of bone regenerative medicine which aims to generate new, cell-driven, functional tissues. In essence, bone-tissue engineering combines progenitor cells, such as human Mesenchymal Stem Cells (hMSC) or mature cells (for osteogenesis) seeded in biocompatible scaffolds and ideally in three-dimensional tissue-like structures (for osteoconduction and vascular ingrowth), with appropriate growth factors (for osteoinduction), in order to generate and maintain bone. The need for such improved composite grafts is obvious, especially for the management of large bone defects, for which the requirements for grafting material are substantial. One of the main disadvantages of tissue-engineering approaches is their substantial cost, mainly due to use of recombinant proteins (Growth factors like Bone Morphogenetic Proteins). Therefore identification of molecules that can enhance bone formation is highly desirable.
EP 0956865 A1 discloses numerous Rho kinase inhibitors that have various therapeutic effects. In Exp. Example 8 some of the compounds have shown to have inhibitory action on bone resorption in vitro. Meanwhile, none of these tested compounds share the chemical structure of the compounds of the present invention. EP 0956865 A1 has a general formula (II), which encompasses the compounds of the present invention. According to the description in EP 0956865 A1 the compounds of formula (II) can be used to treat hypertension, angina pectoris, cerebrovascular contraction, asthma, inflammation and, brain function disorder, eripheral circulation disorder, arteriosclerosis, cancer, autoimmune diseases, AIDS, osteoporosis, retinopathy, immature birth, and digestive tract infections. However, there is no specific teaching in EP 0956865 A1 that the claimed compounds of the present invention would be good enhancers of bone formation.
Soerensen et al (BMC Muscular Disorders, 2010, vol 11, no 250, pp 1471-1474) disclose tests of kinase inhibitors in osteoclast and there effect on bone resorption. One of the inhibitors tested is H-8 (table 1) that falls under the claimed subject-matter of the present invention. Meanwhile, according to table 1 in Soerensen et al H-8 has no effect on osteoclastic acid secretion. Thus, Soerensen et al teaches away from the present invention.